Methods of predicting therapeutic efficacy of treatment of a multiple sclerosis patient

ABSTRACT

Human myelin basic protein (h-MBP) has a molecular weight of 18.5 KD and contains 170 amino acid residues. Synthetic peptides ranging in length from about 8 to 25 residues and covering the entire length of the protein have been produced. Antibodies to h-MBP (anti-MBP) were found to be neutralized by the synthetic peptides, in vitro, which span the h-MBP from about amino acid residue 61 to about amino acid residue 106. The peptides, which cover both the amino (about residues 1 to 63) and carboxy (about residues 117 to 162) terminals of h-MBP did not neutralize purified anti-MBP. Intrathecal administratin of peptide MBP(75-95), MBP(86-95), or MBP(82-98) produced complete binding-neutralization of free (F) anti-MBP with no change in bound (B) levels. A control peptide MBP35-58 had no effect on F or B anti-MBP levels. Intravenous administration of MBP(75-95), MBP(86-95), or MBP(82-98) resulted in significant decline of F and B CSF anti-MBP levels. Administration of MBP synthetic peptides to MS patients either intrathecally or intravenously did not have any adverse neurological effects and systemic complications did not occur. The MBP epitope for MS anti-MBP has been localized to an area between amino acid 86 and amino acid 95.

FIELD OF INVENTION

This invention is concerned with selected polypeptides and their use inthe immunoregulation of antibodies to human myelin basic protein. Thisinvention also relates to novel pharmaceutical compositions containingthese selected polypeptides and to a method of using these peptides forthe treatment of Multiple Sclerosis.

BACKGROUND AND PRIOR ART

Multiple sclerosis (MS) is a multifocal demyelinating disease of thehuman central nervous system (CNS) associated with inflammation.Increased intra-blood-brain barrier (intra-BBB) IgG synthesis is ahallmark of MS (Tourtelotte, W. W., J Neurol Sci 10: 279-304, 1970;Link, H. and Tibbling, G., Scand J Clin Lab Invest 37: 397-401, 1977;Tourtelotte, W. W. and Ma, B., Neurology 28: 76-83, 1978; Walsh, J. M.and Tourtelotte, W. W., In: Hallpike, J. F., Adams, C. W. M. andTourtelotte, W. W., eds. Multiple sclerosis. Baltimore. Williams &Wilkins, 1982: 275-358; and Warren, K. G., and Catz, I. Ann Neurol 17:475-480, 1985).

IgG synthesis within the BBB is generally elevated in clinicallydefinite MS patients (Schumacher, G. A., Beebe, G., Kibler R. E., etal., Ann NY Acad Sci 15:266-272, 1965) with active or inactive disease.The specificity of the majority of the CNS IgG is unknown. While a smallproportion has antiviral activity or reacts against brain antigens,nucleic acids, erythrocytes or smooth muscle antigens, the nonspecificportion may represent polyclonal activation of B-cells (Tourtelotte, W.W., and Ma, B., Neurology 28:76-83, 1978). During the last decade therehas been considerable interest in the study of antibodies to specificmyelin proteins.

Following the detection of circulating immune complexes containingmyelin basic protein (MBP) as their antigenic component (Dasgupta, M.K., Catz, I, Warren, K. G. et al., Can J Neurol Sci 10:239-243, 1983),increased titers of antibodies to MBP (anti-MBP) were observed in thecerebrospinal fluid (CSF) of patients with active forms of MS (Warren,K. G. and Catz, I., Ann Neurol 209:20-25, 1986). Clinically, MS ischaracterized by phases of disease activity such as acute relapses orchronic progression, and by phases of clinical remission. Active MS isassociated with increased levels of intrathecally produced anti-MBP(Warren, K. G. and Catz, I., Ann Neurol 209:20-25, 1986; and Catz, I.and Warren, K. G., Can J Neurol Sci 13:21-24, 1986). These antibodiesare found predominantly in free (F) form during acute relapses andpredominantly in bound (B) form when the disease is insidiouslyprogressive (Warren, K. G. and Catz, I., Ann Neurol 209:20-25, 1986).During acute relapses, CSF anti-MBP titers correlated with diseaseactivity (Warren, K. G. and Catz, I., Ann Neurol 21:183-187, 1987).Anti-MBP levels were also increased in patients with first attacks ofoptic neuritis and in most patients experiencing first attacks of MS(Warren, K. G., Catz, I., and Bauer, C., Ann Neurol 23:297-299, 1988;Warren, K. G. and Catz, I., J Neurol Sci 91:143-151, 1989).

Longitudinal kinetic studies of CSF anti-MBP levels in patients whoenter the recovery phase subsequent to an acute relapse, demonstrated agradual decline in F anti-MBP titers commensurate with a progressiverise in B fractions (Warren, K. G. and Catz, I., J Neurol Sci91:143-151, 1989; Warren, K. G. and Catz, I., J Neurol Sci 88:185-194,1988). In the remission phase, CSF anti-MBP may become undetectablesuggesting an anti-MBP neutralization associated with inactive phases ofMS (Warren, K. G. and Catz, I., J Neurol Sci 88:185-194, 1988). Incontrast, chronic-progressive MS characterized by persistence ofincreased anti-MBP over long periods of time was associated withinhibition of anti-MBP neutralization (Warren, K. G. and Catz, I., JNeurol Sci 88:185-194, 1988). Recently a myelin basic protein antibodycascade, identified in the IgG fraction purified from CSF of MSpatients, contained anti-MBP, antibodies which neutralize anti-MBP andantibodies which inhibit anti-MBP neutralization (Warren, K. G. andCatz, I., J Neurol Sci 96:19-27, 1990).

Our previous research has demonstrated from the B-cell autoimmune pointof view that there are at least two distinct forms of MS with themajority of patients having autoantibodies to myelin basic protein(anti-MBP) and a lesser number having antibodies to proteolipid protein(anti-PLP) (Warren, K. G. et al., Ann. Neurol. 35, 280-289, 1994). Inanti-MBP associated MS, acute relapses are associated with elevated(greater than 1) Free (F)/Bound (B) anti-MBP ratios whereas the chronicprogressive phase is characterized by F/B anti-MBP ratios of equal orless than 1, and patients in remission sometimes have mildly elevated Banti-MBP titers (Warren, K. G. and Catz, I., J. Neurol. Sci. 88,185-194, 1989).

It has been demonstrated that some of the proliferating T-cells in MSpatients are directed towards MBP (Allegretta et al., Science, 247,718-721, 1990) and that human T-cells can recognize multiple epitopes onthe molecule (Richert et al., J. Neuroimmun 23, 55-66, 1989). MBP alsoappears to be capable of activating some T-cells without the involvementof antigen presenting cells (Altman et al., Eur. J. Immun. 17,1635-1640, 1987). It is likely that small peptides of MBP may berecognized by T-cells without the requirement for intracellularprocessing, simply by their ability to bind class II majorhistocompatibility antigens on the surface of presenting cells.

Since experimental allergic encephalomyelitis (EAE), an accepted animalmodel of MS, can be induced by inoculating susceptible rodents witheither MBP or PLP in conjunction with Freund's complete adjuvant, theprocess of MS demyelination may have an autoimmune mechanism (Fritz, R.B. et al., J. Immunol. 130, 1024-1026, 1983; Trotter, J. L. et al., J.Neurol. Sci. 79, 173-188, 1987). From B-cell autoantibody point of view,the MBP epitope targeted by the disease process has been localizedproximal to the tri-Prolil sequence (residues -99-100-101-) to an areabetween residues 80 and 100 (Warren, K. G. et al., Ann. Neurol. 35,280-289, 1994). This B-cell epitope overlaps the immunodominant epitopefor T cells reactive to MBP, which are found in MS brain lesions(Oksenberg, J. R. et al., Nature, 362, 68-70, 1993).

Previous studies have shown that anti-MBP is neutralized by MBP.However, previous attempts to treat MS by intramuscular or subcutaneousadministration of heterologous MBP have not been successful (Campbell,B., Vogel, R. J., Fisher, E. and Lorenz, R., Arch Neurol 29:10-15, 1973;Gonsette, R. E., Delmotte, P. and Demonty, L., J Neurol 216:27-31, 1977;and Romine, J. S. and Salk, J., In: Hallpike, J. F., Adams, C. W. M. andTourtelotte, W. W., eds. Multiple sclerosis. Baltimore, Williams &Wilkins, 1982:621-630). The problem with using native MBP is two-fold.Firstly, the protein is prepared from human brain samples andaccordingly there is a potential danger that latent neuroviruses may bepresent in the sample. Secondly, although soluble MBP is not usually animmunogen, it is possible that when administered to individuals with analtered immune system, soluble MBP could act as an antigen and cause theproduction of antibodies against MBP.

Accordingly, the present invention determines whether anti-MBP purifiedfrom CSF of MS patients can be neutralized by selected soluble peptidesof human MBP (h-MBP). For this purpose, soluble synthetic peptidescovering the entire length of h-MBP were used to determine the possibleepitope range on h-MBP which neutralizes anti-MBP obtained from thesepatients. Therefore selected soluble peptides, which demonstrateneutralization of anti-MBP, can be used to treat MS more effectivelythan the whole molecule. These soluble peptides are syntheticallyproduced and as such no potential threat of neuroviruses would exist.Additionally, due to their small size, these peptides could not act asan immunogen. Therefore, the use of selected peptides as a treatment forMS, would overcome the problems identified with using the nativeprotein.

Further the peptides of the present invention were investigated todetermine their effectiveness in binding or modulating the production ofMS anti-MBP in vivo.

SUMMARY OF INVENTION

According to the present invention there is provided, peptides which aresubstantially homologous in sequence to a part of the amino acidsequence of a human myelin basic protein. These peptides are capable ofneutralizing or modulating the production of anti-MBP.

According to the present invention the peptides are of the formula:R₁-Val-His-Phe-Phe-Lys-Asn-IIe-R₂and salts thereof, wherein R₁ and R₂ are independently selected from thegroup consisting of hydrogen, hydroxy, the residue of an amino acid andthe residue of a polypeptide; provided that R₁ and R₂ are not bothhydrogen or hydroxyl at the same time. The peptide can containsubstitutions, deletions or additions thereof, provided that the peptidemaintains its function of neutralizing or modulating the production ofanti-MBP.

Examples of said peptides are selected from: MBP75-95 Lys Ser His GlyArg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val ThrMBP64-78 Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gln Lys Ser His GlyMBP61-75 His His Pro Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gln LysMIBP69-83 Tyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp GluMBP80-97 Thr Gln Asp GIu Asn Pro Val Val His Phe Phe Lys Asn Ile Val ThrPro Arg MBP91-106 Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser GlnGly Lys Gly MBP84-93 Asn Pro Val Val His Phe Phe Lys Asn Ile MBP85-94Pro Val Val His Phe Phe Lys Asn Ile Val MBP86-95 Val Val His Phe Phe LysAsn Ile Val Thr MBP87-96 Val His Phe Phe Lys Asn Ile Val Thr ProMBP82-98 Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro ArgThr

Further according to the present invention there is providedpharmaceutical compositions, which comprises as an active ingredient apeptide as described above, either alone or in combination, in admixturewith a pharmaceutical acceptable carrier.

Further according to the present invention, there is provided a methodof treating multiple sclerosis comprising administering an effectiveamount of a peptide as, described above, either alone or in combinationto effectively neutralize or modulate the production of anti-humanmyelin basic protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the localization of eighteen synthetic peptides (smallnumbers) in relation to the intact human-MBP molecule. Peptides arerepresented by vertical bars placed next to their corresponding regionon the MBP molecule. Large numbers represent amino acid residues onhuman MBP.

FIG. 2 shows inhibition curves of anti-MBP, purified and pooled from 10different multiple sclerosis patients, by human MBP and MBP-peptides.

FIG. 3 shows the neutralization of anti-MBP isolated from an individualmultiple sclerosis patient by human MBP and peptides MBP80-97; MBP91-106and MBP75-95.

FIG. 4—Longitudinal monitoring of CSF anti-MBP titers in a patient withchronic progressive MS:

F (Free) and B (Bound) levels of anti-MBP were persistently elevatedwhen sampled 26 times over a period of 11 years from 1983 to 1993.

-   cpm: counts per minute    ${{radioactivity}\quad{units}} = \frac{{{cpm}\quad{sample}} - {{cpm}\quad{blank}}}{{{cpm}\quad{total}} - {{cpm}\quad{blank}}}$-   open circles: Bound (B) anti-MBP determined after acid hydrolysis of    CSF immune complexes.-   closed circles: Free (F) anti-MBP

FIG. 5—Control patients: CSF anti-MBP levels in 2 “time controls” (1F56,FIGS. 5A and 3M66, FIG. 5B) and 2 “time-saline controls” (4M45, FIGS. 5Cand 5M59, FIG. 5D). In all four patients F and B anti-MBP remainedconstantly elevated at baseline level when CSF was sampled every 30minutes for the first two hours as well as 24 hours later. Symbols as inFIG. 4.

FIG. 6—Interpatient peptide studies: CSF anti-MBP levels in a group offour patients (10F38, FIG. 6A; 13F43, FIG. 6C; 5M59, FIG. 6D; and 3M66,FIG. 6G) who received increasing amounts (1, 2.5, 5 and 10 mgrespectively) of a non-binding, control synthetic peptide MBP35-58 and apaired group of four other MS patients (6F53, FIG. 6B; 8M41, FIG. 6D;4M45, FIG. 6F; and 1F56, FIG. 6H) who received increasing amounts (1,2.5, 5 and 10 mg respectively) of the anti-MBP binding synthetic peptideMBP75-95. CSF F anti-MBP was bound in a dose-dependent fashion bypeptide MBP75-95 and it did not react with peptide MBP35-58. Boundanti-MBP remained virtually unaffected.

FIG. 7—Intrapatient peptide studies: when MS patients were either “timecontrols” (1F56, FIGS. 7C and 3M66, FIG. 7D) or “time-saline controls”(5M59, FIGS. 7A and 4M45, FIG. 7B), or when they received thenon-binding, control peptide MBP35-58 (5M59 and 3M66) their F and B CSFanti-MBP levels remained unaffected. In contrast, when the same patients4M45, 1F56 and 3M66 later received 5-10 mg of the anti-MBP bindingpeptide MBP75-95, their F anti-MBP became undetectable for periods up to7 days and returned to baseline level between 10 and 21 days.

FIG. 8—Repeated intrathecal synthetic peptide injections: a patient withchronic progressive MS received 10 weekly injections of 10 mg MBP75-95inoculated directly into the CSF; F and B titers of anti-MBP weremeasured before (circles) and 30 minutes after (squares) eachinoculation. F anti-MBP (closed circles and squares) was renderedundetectable for the 10 week period while B antibody remainedessentially unchanged (open circles and squares).

FIG. 9—Intravenous synthetic peptide administration: CSF anti-MBP levelsfollowing a single intravenous injection of 500 mg MBP75-95; both F andB anti-MBP levels declined significantly when tested 10, 16 and 30 daysafter injection. Symbols as in FIG. 4.

FIG. 10. Further refinement of the MBP epitope for MS anti-MBP using aset of 41 decapeptides which covered the area between residues 61 and110.

Legend:

-   -   bars represent percent inhibition=100−radioactivity units    -   MBP and peptide MBP75-95 were used as positive controls and        produced complete (100%) inhibition of both F and B antibody    -   peptides MBP51-60 and MBP 111-120 were used as negative controls        and produced insignificant inhibition (0-10%) of F and B        anti-MBP    -   decapeptides MBP84-93, MBP85-94, MBP86-95 and MBP87-96 which        produced maximum inhibition (90-100%) of both F and B antibody        are highly associated with the MBP epitope    -   dotted line: 95% confidence limits of the inhibition assay

FIG. 11 a shows free (F)-● and bound (B)-∘ CSF anti-MBP in a patientwith unilateral optic neuritis who received intrathecally two injections(it#1 and it#2) of 50 mg pMPB86-95, 4 weeks apart; w=number of weeks.

FIG. 11 b shows free (F)-● and bound (B)-∘ CSF anti-MBP levels in apatient with complete unilateral optic neuritis who received multipleintrathecal injections (it#1, it#2, it#3, it#4 and it#5) of 50 mgpMBP82-98 during the first week of relapse.

FIG. 11 c shows free (F)-● and bound (B)-∘ CSF anti-MBP levels in apatient with pseudoatherosis who received five daily intrathecalinjections (it#1, it#2, it#3, it#4 and it#5) of 50 mg pMBP82-98.

FIG. 11 d shows free (F)-● and bound (B)-∘ CSF anti-MBP levels in apatient with relapsing-progressive MS who received four intrathecalinjections (it#1, it#2, it#3 and it#4) of 50 mg pMPB86-95 every 2 to 3days during the first week of a relapse and one intravenous injection(IV) of 400 mg pMPB86-95 when the disease reentered the progressivephase.

FIG. 12 shows free and bound CSF anti-MBP levels in a patient with apolysymptomatic relapse who received a total of seven intrathecalinjections of 50 mg pMPB86-95. No CSF sample was obtained 30 minutesafter it#2; a CSF sample was obtained 24 hours later. Symbols as in FIG.11.

FIG. 13 shows free and bound CSF anti-MBP levels in a patient withrelapsing-progressive MS who received both intrathecal (it#1, it#2 andit#3) and intravenous (IV#1 and IV#2) injections of pMPB86-95. No CSFsample was obtained before or after it#2. Symbols as in FIG. 11.

FIG. 14 shows free and bound CSF anti-MBP levels in a patient withrelapsing-progressive MS who received intravenous (IV#1, IV#2 and IV#3)and intrathecal (it#1 to it#9) injection of pMPB86-95 and pMPB82-98.Symbols as in FIG. 11.

FIG. 15 shows the attempt to prevent future relapses in a patient withrelapsing-progressive MS who received two intravenous injections (IV#1and IV#2) of 400 mg pMPB86-95 and pMPB82-98. No CSF sample was obtainedduring the first relapse, 3 months after IV#1. Natural rate of relapsesis represented at the top by arrows corresponding to the month of theattack. Boxed area represents the time of the experiment. Symbols as inFIG. 11.

FIG. 16 hows the attempt to prevent future relapses in a patient withrelapsing-progressive MS who received two intrathecal (it#1 and it#2)and one intravenous injection (IV) of pMPB86-95. ▪, high dose ofintravenous methylprednisolone. Natural rate of relapses is representedat the top by arrows corresponding to the month of the attack. Boxedarea represents the time of the experiment. Symbols as in FIG. 11.

FIG. 17 shows the effect of intrathecal and intravenous peptideadministration of MBP specific autoantibodies in CSF of a chronicprogressive MS patient; wherein in FIG. 17 a pMBP75-95 was injecteddirectly into CSF (2.5 mg in 5 ml of saline) and MBP specificautoantibodies were measured by a solid-phase radioimmunoassay atdifferent time points (0.5 hours to 7 days following injection). Peptideinjection resulted in transient neutralization of free anti-MBP (closedcircles) but did not affect bound anti-MBP (open circles).Autoantibodies were undetectable at 1 and 2 hours and started to returnto baseline values between 12 and 24 hours following injection. Similarobservations were made in seven other chronic progressive MS patients.In FIG. 17 b, thirteen months following intrathecal peptide injectionshown in FIG. 17 a, 500 mg of pMBP75-95 were injected intravenously in50 ml of saline and MBP specific autoantibodies in CSF were measuredover a 3 month period (mean±standard deviation).

FIG. 18 shows a composite of CSF anti-MBP levels in thirteen patientswith chronic progressive MS who were given an intravenous injection(IV#1 of 5 to 6 mg/kg body weight (256-500 mg in normal saline) ofpMBP75-95 (2 patients) or pMBP86-95 (11 patients); both free and boundanti-MBP (closed and open circles, respectively) were determined.Autoantibody levels were low or undetectable between one and four monthsafter IV#1, when they started to return to baseline levels. Between 6and 10 months after IV#1, all patients received a second intravenousinjection of pMBP82-98 at the same dose (IV#2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to selected peptides, which aresubstantially homologous in sequence to a part of the amino acidsequence of a human myelin basic protein. By ‘substantially homologous’it is meant that some variation between the amino acid sequence of humanmyelin basic protein and the peptides can exist provided that thepeptides, with a variation in amino acid sequence, still function intheir intended use, i.e. to down regulate the production of antibodiesto human myelin basic protein (anti-MBP). Given the teachings of thepresent invention, it would be readily apparent to persons skilled inthe art to determine, empirically, what variation can be made to thesequence of the selected peptides without affecting the function of thepeptides.

Based on further work related to the present invention, on the basis ofthe competitive inhibition assays using a series of 41 decapeptides, theMBP epitope for MS anti-MBP has been refined and localized to an areabetween amino acid 86 and amino acid 95. Based on the highest level ofinhibition, (equal or greater than 95%) of B-anti MBP, the MBP epitopefor MS anti-MBP is between amino acid 86 and amino acid 95. The smallestcommon region of the effective decapeptides is from amino acid 87 toamino acid 93. Thus, according to the present invention, the peptidescan be illustrated by the following formula:R₁-Val-His-Phe-Phe-Lys-Asn-Ile-R₂and salts thereof, wherein R₁ and R₂ are independently selected from thegroup consisting of hydrogen, hydroxy, the residue of an amino acid andthe residue of a polypeptide; provided that R₁ and R₂ are not bothhydrogen or hydroxyl at the same time.

The 7 amino acids spanning amino acid position 87 to 93 would probablynot be large enough to effectively bind anti-MBP. Thus, R₁ and R₂ cannotboth be hydrogen or both be hydroxy at the same time.

When R₁ or R₂ is an amino acid, the amino acid can be selected fromnaturally occurring amino acids. R₁ or R₂ are not restricted to theamino acids occurring upstream or downstream of Val87 and Ile93 in thehuman myelin basic protein, as shown in SEQ ID NO: 1. Variousmodifications, including substitutions, additions or deletions in theupstream and downstream sequences of R₁ and R₂ can be used. In addition,modification, including substitutions, additions or deletions can bemade to the sequence -Val-His-Phe-Phe-Lys-Asn-Ile, provided that thepeptides so produced still function in their intended use; i.e., toneutralize or modulate the production of antibodies to myelin basicprotein.

The term “residue of polypeptide” or “polypeptide residue” is meant toinclude different size polypeptides including proteins or fragmentsthereof. As above, when R₁ or R₂ is a polypeptide residue, R₁ or R₂ arenot limited to the peptides occurring upstream or downstream of Val87and Ile93, in the human myelin basic protein. Any polypeptide residuecan be used.

In one embodiment of the invention R₁ can be a peptide selected from thegroup of peptides ranging from animo acid residue 61 to amino acidresidue 86 of SEQ ID NO:1. The length of said peptide can range from asingle amino acid residue to a 26 amino acid residue.

In a further embodiment of the present invention R₂ can be a peptideselected from the group of peptides ranging from animo acid residue 94to amino acid residue 106 of SEQ ID NO:1. The length of said peptide canrange from a single amino acid residue to a 13 amino acid residue.

R₁ and/or R₂ could be a repeat of the sequence-Val-His-Phe-Phe-Lys-Asn-Ile, or modifications thereof, includingsubstitutions, additions or deletions. Thus, the peptide could containmultiple copies of the anti-MBP binding site (epitope).

The compounds of the present invention can be prepared according towidely acceptable methods of synthesizing polypeptides. Also includedwithin the scope of the term ‘peptide’ are peptides produced byrecombinant DNA technology. Knowing the sequence of the selectedpeptides, as disclosed in the present invention, it is within the scopeof the present invention to determine an appropriate DNA sequence, whichwill code for the selected amino acid sequence. The appropriate DNAsequence can be produced by conventional, known methods of synthesizingDNA sequences. The DNA sequences so produced can then be cloned intoappropriate cloning vehicles and used to transform an appropriate hostcell to produce the recombinant peptide. All of the methodology referredto above is conventional and well-known to persons skilled in the art.

The peptides, of the present invention, are substantially homologous insequence to a part of the amino acid sequence of human myelin basicprotein. By ‘a part of the amino acid sequence’ it is meant that thesequence can be of any length provided that the amino acid sequence islong enough to function to down regulate production of anti-human myelinbasic protein but not of a length which would result in prior artproblems when MBP peptides were used for in vivo treatment of MultipleSclerosis. According to the present invention the peptides can be atleast 10 amino acids in length. In one example of the present inventionthe peptides can be from about 10 amino acid residues to about 25 aminoacid residues. If the peptides of the present invention are used as partof a fusion protein, the overall size of the peptide can be much larger.

According to one embodiment of the present invention it has beendetermined that selected peptides substantially corresponding to theamino acid sequence of h-MBP are effective in down regulating productionof anti-MBP. These peptides correspond to the amino acid sequence ofh-MBP from about residue 61 to about 106. In one example these peptidescorrespond to the amino acid sequence of the h-MBP from residue 75 toabout residue 106, when the peptides are used for the neutralization offree anti-MBP. In a further example, these peptides correspond to theamino acid sequence of the h-MBP from about residue 82 to about residue99, when the peptides are used for neutralization of F anti-MBP or downregulation of synthesis of F and B anti-MBP. Therefore the peptides areselected from 10 amino acid residues to 25 amino acid residues takenfrom a continuous amino acid sequence within the sequence shown below(SEQ ID NO: 1), provided that said sequence can neutralize or modulatethe production of the anti-myelin basic protein. SEQ ID NO: 1 61 His HisPro Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg ThrGln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg ThrPro Pro Pro Ser Gln Gly Lys Gly                   106

Examples of peptides are selected from the group consisting of: MBP61-73His His Pro Ala Arg Thr Ala His Tyr Gly Ser Leu Pro Gln Lys MBP64-78 AlaArg Thr Ala His Tyr Gly Ser Leu Pro Gln Lys Ser His Gly MBP69-83 Tyr GlySer Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu MBP75-95 Lys Ser HisGly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val ThrMBP80-97 Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val ThrPro Arg MEP91-106 Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser GlnGly Lys Gly

In one embodiment of the present invention, the peptides are representedby the formula: R₁-Val-His-Phe-Phe-Lys-Asn-Ile-R₂and salts thereof, wherein R₁ and R₂ are independently selected from thegroup consisting of hydrogen, hydroxy, the residue of an amino acid andthe residue of a polypeptide; provided that R₁ and R₂ are not bothhydrogen or hydroxyl at the same time. The peptide can containsubstitutions, deletions or additions thereof, provided that the peptidemaintains its function of neutralizing or modulating the production ofanti-MBP.

Examples of peptides are selected from: MBP84-93 Asn Pro Val Val His PhePhe Lys Asn Ile MBP85-94 Pro Val Val His Phe Phe Lys Asn Ile ValMBP86-95 Val Val His Phe Phe Lys Asn Ile Val Thr MBP87-96 Val His PhePhe Lys Asn Ile Val Thr Pro MBP82-98 Asp Glu Asn Pro Val Val His Phe PheLys Asn Ile Val Thr Pro Arg Thr

The peptide MBP82-98 has an improved solubility over the other peptidesused in the present invention, due to the five additional hydrophilicresidues in this peptide. Thus, the use of this peptide is preferredover the other peptides disclosed in the present invention.

The potential role of anti-MBP in the pathogenesis of MS continues to beexplored. Increased anti-MBP titers in patients with active MS wereinitially reported by Panitch et al (Panitch, H. S., Hooper, C. S., andJohnson, K. P., Arch Neurol 37:206-209, 1980) who used a solid phaseradioimmunoassay with guinea-pig MBP. Patients with acute MS relapseshave usually increased anti-MBP predominantly in free form, while somepatients in clinical remission may have undetectable anti-MBP levels.During the transition phase from an acute relapse to remission, titersof free anti-MBP progressively decrease over weeks or months, whilebound fractions of the antibody rise as compared to their initial value.In other patients in remission, it is possible to observe low titers offree and bound anti-MBP, usually with a F/B ratio below unity,suggesting that anti-MBP neutralizing antibody(ies) are bound toanti-MBP. Occasionally, patients who fit the criteria of clinicallydefinite MS or patients who had neuropathologically confirmed MS hadundetectable anti-MBP during active phases of their disease. It ispossible that such patients have antibodies to other myelin proteins.The absence of a specific antibody scenario does not negate thepotential importance of anti-MBP in the mechanism of demyelination inthe majority of MS patients.

Recently, an MBP antibody cascade was observed in the IgG fractionpurified from MS CSF (Warren, K. G. and Catz, I., J Neurol Sci 96:19-27,1990). Primary antibodies to MBP in both free and bound forms occur inassociation with active disease: F/B ratios are above unity in patientswith acute relapses, and below unity in patients with chronicprogressive disease (Warren, K. G. and Catz, I., Ann Neurol 209:20-25,1986; Catz, I. and Warren, K. G., Can J Neurol Sci 13:21-24, 1986; andWarren, K. G. and Catz, I., Ann Neurol 21:183-187, 1987). Secondaryantibodies which neutralize anti-MBP appear when the disease becomesinactive. Tertiary antibodies which inhibit anti-MBP neutralization arepresent when the disease is chronically progressive and fails to becomeinactive. The fact that an MBP antibody cascade is associated withdistinct phases of MS suggests its possible importance vis-a-vie thenatural history of this illness.

Although anti-MBP can be detected in CSF of patients with active MS,their direct role in the pathogenesis of demyelination remains to beconfirmed. The involvement of anti-MBP in the mechanism of MS could bestbe determined by their down regulation, in vivo, perhaps byadministration of selected peptides and monitoring the clinical courseof the disease. If anti-MBP is (are) the only primary antibody(ies)associated with demyelination in MS, it may be possible to block thisprocess by intrathecal, and/or intravenous administration, of selectedMBP peptides which would down regulate anti-MBP and would promotetolerance to MBP in situ. Other human myelin proteins may also beinvolved with the demyelination in MS and accordingly, it is within thescope of the present invention to use peptides substantially homologousin sequence to a part of the amino acid sequence of these other myelinproteins to down regulate the corresponding antibodies. Althoughprevious attempts to treat MS by intramuscular or subcutaneousadministration of heterologous MBP have not been entirely successful(Campbell, B., Vogel, R. J., Fisher, E. and Lorenz, R., Arch Neurol29:10-15, 1973; Gonsette, R. E., Delmotte, P. and Demonty, L. J Neurol216:27-31, 1977; and Romine, J. S. and Salk, J., In: Hallpike, J. F.,Adams, C. W. M. and Tourtelotte, W. W., eds. Multiple sclerosis.Baltimore. Williams & Wilkins, 1982:621-630), intrathecal and/orintravenous administration of MBP peptides which neutralize or downregulate the production of anti-MBP, according to the present invention,has demonstrated more beneficial results.

The animal model of MS, experimental allergic encephalomyelitis (EAE),is a T cell mediated demyelinating disease. EAE can be ameliorated byintraperitonial inoculation of affected mice with MBP synthetic peptides(Gaur, A. et al., Science 258, 1491-1494, 1992). Furthermore,administration of high doses MBP peptides deleted autoreactive T cellsand abrogated clinical and pathological signs of EAE in mice(Critchfield, J. M. et al., Science 263, 1139-1143, 1994). Even oraladministration of MBP modulated EAE by inducing peripheral tolerance(Chen, W. et al., Science. 265, 1237-1240, 1194). A combination ofmyelin antigens or synthetic peptides of these antigens administered byintravenous and/or intrathecal routes may be required to modulate the Tcells, B cells and macrophages involved in the destruction of myelin inMS patients.

Accordingly, this invention also relates to pharmaceutical compositionscontaining as an active ingredient a peptide as described above, eitheralone or in combination, in admixture with a pharmaceutical acceptablecarrier. Examples of pharmaceutical acceptable carriers are well knownin the art, and include for example normal saline.

The peptides of the present invention can be administered to humans forthe treatment or modulation of Multiple Sclerosis. The therapeutic dose,for intravenous administration, for the treatment of MS may be fromabout 1.0 mg per kilogram of body weight to about 10.0 mg per kilogramof body weight; for intrathecal administration, the total dose may befrom about 1 to about 100 mg. In one example of the present invention,the peptide is administered either intravenously or intrathecally, or incombination. The peptides can be administered as a single or sequentialdose, as may be required.

According to the present invention intravenous administration was foundto down regulate both free and bound anti-MBP; whereas, intrathecaladministration was only effective in neutralizing or modulating freeanti=MBP.

In one embodiment of the present invention it was found that sequentialintrathecal administration of MBP peptides, could reduce F anti-MBP, andmaintain its low levels for months after the peptides were injected inpatients suffering from monosymptomatic relapses. In one example of thisembodiment. 50 mg of a peptide of MBP was administered to a patientdaily for 4 to 5 days. In yet a further example a further dose can beadministered one week to two weeks following the initial injections.

While this invention is described in detail with particular reference topreferred embodiments thereof, the following examples, are offered toillustrate but not limit the invention.

EXAMPLE 1 In Vitro Neutralization of anti-Human Myelin Basic Protein

FIG. 1 shows the localization of 18 peptides of h-MBP used in this studyin relation to the intact MBP molecule. Native MBP was isolated fromnon-MS brain tissue (Diebler, G. E., Martenson, R. E., Kies, M. W., PrepBiochem 2:139-165, 1972) and further purified by gel filtration andreverse phase high pressure liquid chromatography (HPLC). The finalantigen preparations were checked for purity by SDS-polyacrylamide gelelectrophoresis. Only preparations that migrated at the molecular weightof 18.5 KD were used in further studies. Purified MBP was used inantigen-specific affinity chromatography, in neutralization studies andin the solid phase anti-MBP radioimmunoassay.

Eighteen peptides covering the length of h-MBP and containing between 8and 25 amino acid residues were synthesized by the Fmoc method aspreviously described (Groome, N. P., Dawkes, A., Barry, R. et al. JNeuroimmun 19:305-315, 1988). Peptide purity was checked byreverse-phase HPLC with a C18 column and water/acetonitrile gradient(0.1% TFA). Amino acid analysis of peptides was also performed usingstandard analysis. Many of the peptides used in this study contained anunnatural cysteine residue as they were made to function as immunogensin conjunction with Freund's adjuvant. This is unlikely to affect thepresent findings.

Cerebrospinal fluid (CSF) was obtained within a week from the onset ofsymptoms from 35 patients with acute MS relapses and IgG levels weredetermined by nephelometry. CSF samples used in this study were selectedto have initially high absolute IgG levels (≧0.080 g/l) and increasedtiters of anti-MBP (F/B ratio>>1.0). All MS patients had clinicallydefinite disease.

IgG was purified from concentrated CSF of patients with acute MS byprotein A-Sepharose (Pharmacia™) affinity chromatography as previouslydescribed (Warren, K. G. and Catz, I., J Neurol Sci 96:19-27, 1990). Thepurity of each IgG preparation was checked by polyacrylamide gelelectrophoresis and isoelectric focusing. When elevated anti-MBP levelsfrom purified IgG were absorbed to zero with MBP, the post-absorptionsupernatants contained residual IgG indicating that anti-MBP representsonly a fraction of the elevated IgG.

Purified MBP was coupled to CNBr-activated Sepharose 4B (Pharmacia™)according to the manufacturer's instructions. Purified CSF IgGcontaining increased anti-MBP levels from 35 patients with acute MSrelapses was used to isolate anti-MBP by MBP-Sepharose affinitychromatography (Warren, K. G. and Catz, I., J Neurol Sci 103:90-96,1991). Purified anti-MBP samples were compared with the initial IgGsource by poly-acrylamide gel electrophoresis. When purified anti-MBPsamples were absorbed to zero with MBP, the post-absorption supernatantscontained no residual IgG indicating the purity of anti-MBP.

Constant amounts of anti-MBP (15 radioactivity binding unitscorresponding to 100 for scale expansion purposes=%0) were incubatedwith increasing amounts of h-MBP (0-1000 ng) or individual peptides ofMBP (0-10,000 ng) in a liquid phase assay and after 1.5 hoursincubation, free anti-MBP levels were determined in all mixtures.Anti-MBP isolated from 7 individual MS patients or pooled anti-MBP from10 different MS patients were used in neutralization experiments. Calfthymus histone and human serum albumin were used as negative antigencontrols (range: 10-1000 ng). One monoclonal antibody (MAb) to peptideMBP64-78 and a polyclonal rabbit antiserum to peptide MBP1-8 were usedas positive antibody controls (Groome, N., Harland, J., and Dawkes, A.,Neurochem Int 7:309-317, 1985; Barry, R., Payton, M., and Groome, N.,Neurochem Int 2:291-300, 1991). Another mouse monoclonal antibody toepitope 45-50 was used as negative antibody control.

Anti-MBP levels were determined by a solid phase radioimmunoassay withhuman MBP (Warren, K. G. and Catz, I., Ann Neurol 209:20-25, 1986;Warren, K. G. and Catz, I., Ann Neurol 21:183-187, 1987; and Warren, K.G. and Catz, I., J Neurol Sci 91:143-151, 1989). Free anti-MBP levelswere measured in all fractions from affinity chromatography and in allneutralization mixtures. All individual samples were run inquadruplicate using the same iodinated material in order to minimizebetween-assay variability.

Purified anti-MBP was completely neutralized by MBP and by peptidesMBP80-97, MBP91-106 and MBP75-95, and was partially neutralized bypeptides MBP64-78, MBP69-83 and MBP61-75 (Table 1 and FIG. 2). Theremaining twelve peptides did not neutralize purified anti-MBP and theirkinetic curves fell within the striped area shown in FIG. 2. Calf thymushistone and human serum albumin did not react with purified anti-MBPeven at concentrations as high as 1000 ng. The MAb to peptide MBP64-78was only inhibited by peptide MBP64-78, and the MAb to peptide MBP 1-8was only inhibited by peptide MBP1-8. The MAb peptide MBP 45-50 did notreact with MBP or any of the peptides (for clarity of the figure,control data are not illustrated). The control samples demonstrate thevalidity of the neutralization approach as each control antibody wasneutralized completely by the expected peptide and by none of the otherpeptides. This shows that even the high peptide concentrations (10,000ng) specificity of recognition was observed. TABLE 1 HUMAN MBPREACTIVITY WITH SEQUENCE ANTI-MBP 1-170 ++ 1-8 Cy − Cy 4-18 − Cy 11-24 −18-32 − 26-40 − Cy 35-58 − Cy 51-64 Gly + Cy 64-78 + Cy 61-75 + Cy 69-83++ Cy 75-95 ++ Cy 80-97 Gly ++ Cy 91-106 − 117-129 − Cy 127-140 − Cy136-149 − 141-155 − Cy 149-162++ complete neutralization+ partial neutralization− insignificant reactivity

Anti-MBP purified from 7 individual MS patients was completelyneutralized by h-MBP and peptides MBP80-97, MBP91-106 and MBP75-95 (seeFIG. 3 as an illustrative example). Due to the limited amount ofantibody obtained from individual MS patients, anti-MBP was not reactedwith the remaining 15 peptides.

As noted previously, anti-MBP was neutralized with peptides spanningfrom about amino acid residue 61 to about amino acid residue 106. Thepeptides which did not neutralize anti-MBP cover both the amino (aboutresidues 1 to 63) and the carboxyl (about residues 117 to 162) terminalsof h-MBP. It appears that peptides from different non-overlappingregions of MBP neutralize the same antibody(ies). This might beexplained if the antibodies recognize a discontinuous (assembled)epitope containing amino acids from different regions. A similarphenomenon has been previously observed by Hruby et al (Hruby, S.,Alvord, E. C., Groome, N. P. et al, Molec Immun 24: 1359-1364, 1987) whoshowed that a rat monoclonal antibody had a major epitope in MBPsequence 112-121 but a strong cross-reaction with another epitope inpeptide 39-91. This is more likely than the possibility that theantibody is cross-reactive with two completely different sequences whichdid not form a discontinuous epitope (Hruby, S., Alvord, E. C.,Martenson, R. E., et al. J Neurochem 44:637-650, 1985). Theneutralization data could be explained by the ability of peptides fromdifferent sections of MBP to each partially occupy the antibody bindingpocket by interacting with different antibody amino acid side chains.This explanation fits the observation that the peptides giving completeinhibition (MBP80-97, MBP91-106 and MBP75-95) are approximately 100times less effective on a molar basis than intact MBP at causinginhibition. By the hypothesis advanced above, this could be due to eachpeptide clone being unable to achieve the binding energy of the originalMBP epitope.

EXAMPLE 2

In Vivo Neutralization or Modulation of Production of anti-Human MyelinBasic Protein

Patient Selection and Control Studies

Patients who participated in this research project were seen in theMultiple Sclerosis Patient Care and Research Clinic of the University ofAlberta, Edmonton, Canada. The patients have been diagnosed as havingclinically definite multiple sclerosis by Schumacher criteria (1965)confirmed by magnetic resonance imaging of the brain and CSFimmunochemistry profiles. In order to illustrate that in chronicprogressive MS anti-MBP was persistently elevated over long periods oftime, months to years, patients had repeated lumbar punctures withmonitoring of F and B anti-MBP. In a patient with chronic progressiveMS, it was observed that the autoantibody remained persistently elevatedfor periods as long as 11 years and that spontaneous decline of anti-MBPlevels did not occur (FIG. 4 is an illustrative example).

In order to determine that initially elevated CSF anti-MBP levelsremained relatively constant over 24 hours, 2 patients (1 F56 and 3M66)had repeated CSF sampling every 30 minutes for 2 hours as well as 24hours later with F and B anti-MBP monitoring (FIGS. 5A and 5B,respectively). Patients 1F56 and 3M66 served as “time controls”. F and Banti-MBP levels remained constantly elevated when CSF was sampled every30 minutes for 2 hours as well as 24 hours later.

In addition the effect of inoculating 5 cc of normal saline into the CSFwas similarly determined in two other patients (4M45 and 5M59; FIGS. 5Cand 5D, respectively). These patients served as “time-saline controls”.When 5 cc of normal saline were injected intrathecally, F and B anti-MBPlevels remained elevated at baseline level when CSF was sampled asabove, thus demonstrating that the “dilution effect” on anti-MBP titerswas negligible.

Anti-MBP levels were determined by a solid phase radioimmunoassay withhuman MBP coated on Immulon microtiter wells. Immulon microtiter wellswere coated with 100 μl of 10 μg/ml of MBP (1 μg/well) and incubatedovernight at 37° C. After quenching with bovine serum albumin (BSA) andthree water washes, the wells were stored at room temperature. Samplesof 100 μl of CSF or tissue extracts diluted to 0.010 gm of IgG/l (with0.01 M Barbitol Buffered Saline (BBS) pH 6.9-7.1, 0.5% BSA and 0.05%Tween 20) were incubated in MBP-coated wells for 1-2 hours at roomtemperature. After 5 buffer washes (with 0.01 M BBS, 0.5 BSA and 0.05%Tween 20), wells were incubated with goat anti-rabbit IgG-Fc specific(in 0.01 M BBS, 0.05% Tween 20, 0.5% BSA) for 1 hour at room temperatureand then rinsed as above. Finally, ¹²⁵I-protein A (or ¹²⁵I-protein G)was added and incubated for 1 hour at room temperature. When¹²⁵I-protein G was used as a tracer, ovalbumin replaced BSA in assaybuffer and for quenching. After three final water washes wells wereindividually counted. Results are expressed in radioactivity defined as:(counts of sample−counts of blank)÷(counts of total radioactivity−countsof blank). All samples are run in 10 replicate and counting time is 10minutes in order to collect >10,000 counts for any positive sample.

Prior to being assayed all CSF and/or tissue samples were diluted to afinal IgG concentration of 0.010 g/l. F anti-MBP was detected directlyin CSF or tissue extract while B levels of antibody were determinedfollowing acid hydrolysis of immune complexes with glycine HCl buffer pH2.2. Non-specific binding was performed for each sample in uncoatedwells. For epitope localization, synthetic peptides were firstly reactedwith purified antibody in a liquid phase competitive assay and thenanti-MBP was determined by radioimmunoassay in all resultingsupernatants. Results of the combined competitive binding assay andradioimmunoassay were expressed as percent inhibition of syntheticpeptide defined as 100−radioactivity units. Samples were done in 10replicates and counted for 10 minutes each in a LKB1275 Minigammacounter. A pool of tissue-purified anti-MBP was used at 5pre-established dilutions as positive controls. Pooled CSF from patientswith non-neurological diseases was used as negative controls. Withinassay reproducibility was between 3 and 5% and between assay variationwas less than 7%.

Persistence of CSF anti-MBP at an elevated and constant level inpatients who participates as controls (time control and diluent control)permitted the next step of this research.

Double Blind Peptide Controlled Phase 1 Experiment—Intrathecal Injection

A Phase 1 experiment to determine the effect of synthetic peptideMBP75-95 on F and B titers of CSF anti-MBP was conducted. Subsequent toreceiving approval from the Research Ethics Board of the University ofAlberta, this project was conducted in patients with clinically definiteMS (Schumacher et al., Ann. N. Y. Acad. Sci., 122, 552-568 1965),severely disabled and with advanced progressive disease. After obtaininginformed consent, 14 patients volunteered for this study; eight patientswere selected on the basis of their initial titre of F CSF anti-MBP(above 8 radioactivity units) (Table 2) to receive one intrathecalinjection of either peptide MBP75-95 which bound anti-MBP in vitro or anon-binding control peptide MBP35-58 (Warren and Catz, 1993b). Theexperiment was conducted in a double blind fashion so that neither theresearchers nor the patients had knowledge of the nature of theinoculum. All peptides were coded with 7 digit randomly generatednumbers by an independent physician. Paired peptides dissolved in 5 ccnormal saline and injected into the CSF by means of a lumbar puncturewere administered in increasing dosages of 1, 2.5, 5 and 10 mg. CSF wassampled prior to injection (baseline), at 30 minute intervals for 2hours after injection, 24 hours later and then at weekly intervals for34 weeks until anti-MBP levels returned to baseline. Cell counts, totalprotein, glucose, IgG and albumin levels were determined in all CSFsamples obtained. F and B anti-MBP levels were determined byradioimmunoassay, as described above. TABLE 2 Disease CSF anti-MBPSelected Patient ID duration (radioactivity units) for #, sex, age(years) Kurtzke EDSS Free(F) Bound(B) research  1F56 10 8.5 - Triplegia9 10 Yes  2M50 18 6 - Paraparesis 2 10 No  3M66 20 9 - Quadriplegia 1112 Yes  4M45 21 9 - Quadriplegia 10 11 Yes  5M59 28 9 - Quadriplegia 810 Yes  6F53 19 9 - Quadriplegia 10 9 Yes  7F33 11 6 - Paraparesis, 5 13No ataxia  8M41 8 8 - Triplegia 9 12 Yes  9M49 7 7 - Paraparesis 5 10 No10F38 7 8.5 - Paraplegia 11 10 Yes 11M49 20 8 - Triplegia 6 13 No 12M3512 6.5 - Paraparesis, 7 12 No ataxia 13F43 15 8 - Paraplegia 9 10 Yes14F32 4 6 - Paraparesis, 8 7 No ataxiaTable 2: Clinical data and CSF anti-MBP levels of 14 patients withchronic progressive MS who volunteered to participate in a Phase 1research study of one intrathecal injection of MBP synthetic peptides.Since an initially high F anti-MBP (>8 radioactivity units) wasnecessary in order to achieve a significant post injection change, only8 of 14 patients were selected for the study.

All peptides used in these studies were synthesized under the “goodmanufacturing product” (GMP) code using the Fmoc (9fluorenylmethoxycarbonyl) method by Procyon Inc. (London, Ontario,Canada).

Peptide purity was checked by reverse phase high pressure liquidchromatography with a C18 column and water-acetonitrile gradientcontaining 0.1% TFA. Mass spectroscopy and aminoacid analysis wereperformed by standard methods. Prior to inoculation all peptides werechecked for pyrogenicity (Vancouver General Hospital, Vancouver,Canada), sterility (Provincial Laboratory for Public Health for NorthernAlberta, Edmonton, Canada) and acute toxicity (Health SciencesLaboratory Animal Services, University of Alberta, Edmonton, Canada) andthey were declared “suitable for administration to humans”. Appropriateamounts of coded synthetic peptides were dissolved in 5 cc of sterilenormal saline (0.9% sodium chloride injection USP, nonpyrogenic, BaxterCorp, Toronto, Canada), filtered two times through 0.22 μm sterilizingfilter units (Millex-GX, Millipore Corp., Bedford, Mass., USA) andadministered into the CSF by means of a lumbar puncture.

Interpatient Peptide Studies

Patients 6F53, 8M41, 4M45 and 1F56 received synthetic peptide MBP75-95capable of binding anti-MBP in vitro and patients 10F36, 13F43, 5M59 and3M66 received a “control”, non-binding synthetic peptide MBP35-58 inincreasing amounts of 1, 2.5, 5 and 10 mg respectively (FIG. 6). Inpatient 6F53 (FIG. 6B) who received 1 mg MBP75-95 a 75% decrease of Fanti-MBP followed by its immediate return to baseline level wasobserved; patient 8M41 (FIG. 6D) who received 2.5 mg MBP75-95 showedcomplete binding-neutralization of F anti-MBP followed by its return tobaseline level within 24 hours; in patient 4M45 (FIG. 6F) who received 5mg MBP75-95, a precipitous and complete F anti-MBPbinding-neutralization occurred and persisted for 7 days, havingreturned to its initial value when sampled 21 days later; patient 1F56(FIG. 6H) received 10 mg MBP75-95 which also produced completebinding-neutralization of F anti-MBP which persisted for 7 days and hadreturned to baseline value when sampled 14 and 28 days later. Boundlevels of anti-MBP were not significantly altered by one intrathecalinoculation of MBP75-95. In patients 10F38, 13F43, 5M59 and 3M66 whoreceived respectively 1, 2.5, 5 and 10 mg of the “control” non-bindingpeptide MBP35-58, F and B levels of CSF anti-MBP remained unchanged frominitially high baseline levels during the 24 hour experiment (FIG. 6A,6C, 6E and 6G, respectively). Traditional CSF parameters of inflammationin MS, such as cell counts, absolute levels of total protein, IgG andalbumin, oligoclonal banding, IgG index and CNS IgG synthesis remainedunchanged prior to and after peptide administration.

Intrapatient Peptide Studies

Intrapatient experiments were conducted in order to minimizeinterpatient variability. In patient 5M59 who was either a“time-control” or received 5 mg of the non-binding peptide MBP35-58, Fanti-MBP levels remained elevated at baseline level during bothexperiments (FIG. 7A). Patient 4M45 was initially a “diluent control”and two months later he received 5 mg MBP75-95. His F anti-MBP remainedconstantly elevated in all samples collected during the “diluent”experiment, and it became undetectable after administration of MBP75-95(FIG. 7B). Similar results were obtained in patient 1F56 who hadpersistently elevated levels of F antibody during a “time control”experiment and after administration of 10 mg MBP75-95 her F anti-MBPbecame undetectable (FIG. 7C). A complete study was performed in patient3M66. His F anti-MBP levels were persistently elevated during a “timecontrol” experiment or when 10 mg MBP35-58 were administered; however,when 10 mg MBP75-95 were injected, F anti-MBP was completely neutralizedand remained undetectable for 7 days (FIG. 7D).

Repeated Administration of Synthetic Peptide MBP75-95

After determining that peptide MBP75-95 neutralized F anti-MBP in vivofor periods in excess of 7 days, it was elected to repeatedly inoculate10 mg MBP75-95 into the spinal fluid at weekly intervals for 10 weeks.This experiment was conducted, in 3 different patients with chronicprogressive MS who have not participated in the single peptide injectionproject and volunteered for this study. F and B anti-MBP were determined1-2 weeks prior to the first inoculation, prior to and 30 minutesfollowing each of the 10 injections and again 1 month after the lastinjection. Cell counts, total protein, glucose, IgG and albumin levelswere determined in all CSFs obtained before each of the 10 injections.Prior to the first and after the last injection blood was obtained andanalyzed for electrolytes, creatinine, cardiac and liver enzymes andhematology panel.

When MS patients with chronic progressive disease received repeatedintrathecal injections of 10 mg MBP75-95 at weekly intervals, forperiods up to 10 weeks, their initially high F anti-MBP was undetectablefor as long as the peptide was administered; when the peptide was nolonger administered, F anti-MBP returned to baseline level within 1month (FIG. 8). Titers of B antibody remained constantly elevatedthroughout the experiment suggesting that in these patients synthesis ofanti-MBP continued and intrathecal peptide administration produced onlya “mopping effect” of F anti-MBP.

Patients who received either a single synthetic peptide injection orrepeated weekly injections had chronic progressive multiple sclerosiswith an advanced degree of neurological disability. None of thesepatients reported worsening of their neurological symptoms or MSexacerbations subsequent to intrathecal peptide administration and acellular response did not develop in CSF. MS patients receiving repeatedinoculations of MBP75-95 have been monitored for systemic complicationsincluding electrolyte changes as well as cardiac-liver-kidneydysfunction and hematology changes and no adverse complications haveoccurred. No adverse effects were observed.

Intravenous Administration of MBP75-95

Subsequent to determining that intrathecal administration of peptideMBP75-95 produced complete binding-neutralization of F anti-MBP with nochange in levels of B antibody, it was decided to determine the effectof intravenous administration of the same peptide on CSF titers of F andB anti-MBP; 500 mg of MBP75-95 were dissolved in 100 cc of normal salineand injected intravenously over 30 minutes into patient 8M41 with CSFanti-MBP monitoring every 30 minutes for the first two hours, 18 hourslater as well as 10, 16 and 30 days later. Blood was obtained beforeinjection as well as 16 and 30 days later and analyzed for electrolytes,creatinine, cardiac and liver enzymes and hematology panel. Spinal fluidwas monitored for cell counts, total protein, glucose, IgG and albuminlevels. No adverse effects were observed.

As shown in FIG. 8, intravenous administration of 500 mg MBP75-95 didnot produce any change in titers of F and B levels of CSF anti-MBPwithin the first two hours. A 30% decline in CSF F anti-MBP was observed18 hours later. When CSF was resampled 10, 16 and 30 days later both Fand B anti-MBP had declined from their initial level of 11 radioactivityunits to 4, 2, and 1 radioactivity units respectively.

A repeated observation in all patients treated intrathecally with MBP75-95 was the persistence of elevated levels of bound antibody, while Fanti-MBP became undetectable in a dose-response fashion. This suggestedthat synthesis of autoantibodies to MBP remained active during andsubsequent to intrathecal administration of MBP75-95. As a consequenceof this observation, MBP75-95 was administered intravenously to apatient who had previously received a single intrathecal injection ofthe peptide. After intravenous administration both F and B levels of CSFanti-MBP showed a significant decline when monitored for periods up toone month. The decline of F as well as B levels of CSF anti-MBPsubsequent to intravenous administration of MBP75-95 suggests that thisroute of administration produced downregulation of the autoimmuneinflammatory process responsible for the synthesis of anti-MBP. In afollow-up study to date anti-MBP levels started to increase 4-6 monthsafter a first intravenous injection; a second intravenous injection ofthe same peptide (booster) produced down regulation of anti-MBPsynthesis for up to 2 years in approximately 70 different patients withchronic progressive MS.

MBP Epitope for MS Anti-MBP

In order to further localize the MBP epitope for MS anti-MBP, F and Banti-MBP purified by affinity chromatography from CSF and MS braintissue (Warren, K. G. et al., Ann. Neurol. 35, 280-289, 1994) werereacted in competitive inhibition assays with 41 consecutive MBPsynthetic peptides of equal length (each of 10 residues and overlappingthe adjacent ones by 9) covering the area between residues 61 and 110 ofhuman MBP. The peptide(s) producing maximum inhibition were consideredto be most highly associated with the antibody binding site.

Maximum inhibition (≧80%) of both purified F and B anti-MBP from MSbrain tissue (FIG. 10) was produced by four decapeptides namelyMBP84-93, MBP85-94, MBP86-95 and MBP87-96 suggesting that the MBPepitope for MS anti-MBP is located between residues 84 and 96. Theminimum area of common amino acid residues is from residue 87 to residue93. B anti-MBP had a more restricted range than F antibody.

The role of anti-MBP antibodies in the pathogenesis of MS demyelinationhas not been elucidated and can only be determined by modulatinganti-MBP in vivo and subsequently observing the clinical andpathological outcomes. For example, during an acute relapse of MS, whenF/B antibody ratios are above unity a peptide known to bind F anti-MBPcould be inoculated intrathecally, in order to bind free circulatingantibody and terminate the clinical effects of the acute relapse; weeklyadministration may be required until remission occurs. In MS patientswith chronic progressive disease and superimposed acute relapses,intrathecal as well as intravenous peptide administration may berequired in order to downregulate inflammatory mechanisms which produceanti-MBP.

EXAMPLE 3 Appropriate Dosage of Intrathecally Administered pMBP86-95 orpMBP82-98 in Acute Relapsing Patients

MS relapses are associated with F/B anti-MBP ratios greater than 1.0 dueto higher levels of free than bound antibody in CSF. Generally, over aperiod of 3 months, as a relapse enters into the subsequentrecovery/remission phase, F anti-MBP levels gradually decline, and whenbiological remission is complete, CSF, F and B anti-MBP generally becomeundetectable in CSF.

Patients who participated in the following Examples had eitherrelapsing-remitting or relapsing-progressive MS.

In this and the following Examples either pMBP86-95 or pMBP82-98 wereused. pMBP86-95 had very low solubility in normal saline since itcontained four hydrophilic and six hydrophobic residues. On the otherhand, pMBP82-98 has increased solubility in normal saline, as a resultof the five additional hydrophilic residues.

Two patients were studied to determine the appropriate dosage ofintrathecally administered pMBP86-95 or pMBP82-98, which will reduceimmediately the F anti-MBP to undetectable levels. One patient had anacute relapse of gait ataxia and truncal dysequilibrium. At the onset ofthe attack, this patient received a single intrathecal injection of 10mg pMBP86-95; F and B anti-MBP levels were measured before and 1 hourafter injection and five more times during the next 3 months. Thisdosage suppressed F anti-MBP only partially and the antibody recoverycurve followed closely the natural course; this patient continued tohave progressive spastic paraparesis and ataxia. It was concluded that asingle intrathecal injection of 10 mg pMBP86-95 was inadequate to fullysuppress F anti-MBP and alter its natural recovery rate.

The other patient, an 18 year old female, with acute optic neuritis whoreceived a single intrathecal injection of 50 mg pMBP86-95, had F and Bantibody levels measured before and 30 minutes after injection. Thirtyminutes after injection F antibody became undetectable. The patientwould not agree to subsequent lumbar punctures. It was thus concludedthat dosages of at least 50 mg are required to bind and neutralize Fanti-MBP in CSF for at least 30 minutes.

EXAMPLE 4

Frequency and Duration of Administration in Patients withMonosymptomatic Relapses

In this example the frequency and duration of administration of pMBPthat would maintain low or undetectable F antibody levels for a longertime period were determined. The four patients studied in this groupreceived synthetic peptides within a week from the onset of an attack.

The first two patients had attacks of acute unilateral optic neuritis.One of these patients (FIG. 11 a) received intrathecally two injectionsof 50 mg pMBP86-95 (it#1, it#2) four weeks apart. After each injection Fanti-MBP became undetectable within 1 h. When measured 1 week after thefirst injection the F anti-MBP was elevated, and 4 weeks later the Fantibody was significantly high. At that time the patient has a secondintrathecal injection (it#2) and F anti-MBP became undetectable after 30minutes but it was not subsequently monitored beyond 24 hours. It wasconcluded that this frequency was inadequate and that multipleinjections during the first week of an attack might be required tomaintain negligible antibody levels.

The second patient with complete unilateral optic neuritis receivedmultiple intrathecal peptide injections of 50 mg pMBP82-98 during thefirst week of his attack: four daily injections (FIG. 11 b: it#1, it#2,it#3, it#4) and a fifth injection (it#5) one week later. The anti-MBPprofile of this patient showed a steady, rapid decline over the 7-dayperiod. More important, 7 weeks and 6 months after it#5, his CSFanti-MBP levels remained undetectable and the patient did not experiencea recurrence of optic neuritis nor any other type of MS relapse. Inaddition the unilateral blindness secondary to optic neuritis recoveredfully.

The same schedule of daily intrathecal injections of 50 mg pMBP82-98 wasthen administered to MS patients with different types ofmono-symptomatic relapses. FIG. 11 c illustrates the anti-MBP profile ofa patient with acute pseudoathetosis of his left hand, who receivedintrathecally five daily injections of 50 mg pMBP82-98 (it#1, it#2,it#3, it#4, it#5) in the second week of his attack. F anti-body levelsdeclined to undetectable values within 4 days and remained undetectablewhen assessed 11 days and one month later. This patient steadilyregained function of his left hand so he could again ride his motorcycleand play the guitar.

The last patient had an attack of acute left hemiplegia superimposed onchronic progressive MS. He had four intrathecal injections of 50 mg ofpMBP86-95 every 2 to 3 days. Anti-MBP was measured before and 30 minutesafter each injection (FIG. 11 d: it#1, it#2, it#3, it#4) and 10 daysafter the first injection. The initially elevated F anti-MBP becameundetectable within 7 days when the patient returned clinically andbiochemically to his initial chronic progressive state, and soonafterwards he received intravenously 400 mg pMBP86-95. This suppressedhis bound antibody level for 4 months after the i.v. injection. However,after his last lumbar puncture at 8.5 months post intravenous injection,the disease had returned to chronic progressive pattern both clinicallyand biochemically.

EXAMPLE 5 Frequency and Duration of Administration in Patients withPolysymptomatic Relapses

The same MBP peptides were then injected in patients withpolysymptomatic attacks, affecting multiple areas of the CNS. This groupconsisted of three patients: one with relapsing-remitting and two withrelapsing-progressive disease.

The first patient had a severe polysymptomatic exacerbation. During thefirst week of the relapse she received three injections of 50 mgpMBP86-95 on days 1, 3 and 7 (FIG. 12: it#1, it#2 and it#3). Anti-MBPwas measured before each injection and 30 minutes later. After receivingthese three injections F anti-MBP was suppressed to almost undetectablelevels. When measured a month later, F anti-MBP was rising and by 1.5months, the relapse was once again clinically active and biochemicallyconfirmed. At that time the patient received a second course of fourintrathecal injections of 50 mg pMBP86-95 on days 45, 48, 49, and 50 ofthe relapse (it #4, it#5, it#6 and it#7). Anti-MBP was measured beforeand thirty minutes after each injection and three more times in thesubsequent two months. Once again F anti-MBP was suppressed for at leasttwo weeks, but the patient relapsed again, and at that time her Fantibody level had returned to the initial pre-relapse level. Clearly amore sustained intrathecal administration of the synthetic peptide, inorder to maintain low/undetectable levels of F anti-MBP for longerperiods of time is required.

The second patient had relapsing-progressive MS (FIG. 13). Initially inthe progressive form (F=B), he received intravenously 500 mg pMBP86-95(IV #1). Although both F and B antibody levels were somewhat decreasedafter one month, 9 weeks after the I.V. injection, the patientexperienced a polysymptomatic clinical relapse associated with a highlyincreased F anti-MBP level. At this time, he received three intrathecalinjections of 50 mg pMBP86-95 (it#1, it#2 and it#3) at days 1, 3 and 12of the relapse, and anti-MBP was measured before, 30 minutes and 24hours after the first and third injection. When examined one monthlater, the patient had returned to his initial clinical and biochemicalstatus of progressive spastic paraparesis when, within 2 weeks, hereceived a second intravenous injection of 500 mg pMBP86-95 (IV#2). Todate F and B CSF anti-MBP levels monitored serially for the next 26months remained suppressed when compared to baseline levels. His abilityto stand and walk improved substantially.

The last patient in this group with MS, initially in the progressivephase (F=B), (FIG. 14), received intravenously 500 mg pMBP86-95 (IV#1).CSF anti-MBP was measured after 9 days, then monthly for 2 months and4.5 months after IV#1. Following this injection, F and B anti-MBP levelswere suppressed for 2 months; 4.5 months after IV#1, the patient wascomplaining of increasing weakness, confirmed clinically as well asbiochemically by increased antibody levels compatible with chronicprogressive disease. Within the next month he received a secondintravenous injection of 500 mg pMBP82-98 (IV#2). CSF analysis of thesample taken just before the second injection, was suggestive of anacute relapse pattern (F>B), and the next day, the patient developedacute diplopia due to a left lateral rectus paresis. At this time he wasclearly experiencing a clinical and biochemical acute relapse, whichpersisted over the next 4.5 months and was characterized by severedysequilibrium of stance and gait, weakness of his legs and doublevision. In an effort to lower his elevated F anti-MBP, this patientreceived intrathecally two courses of pMBP 82-98. During the firstcourse initiated 4.5 months from the beginning of the relapse, hereceived 50 mg pMBP82-98, daily for 5 days (it#1, it#2, it#3, it#4 andit#5) and anti-MBP levels measured before and 30 minutes after eachinjection remained reasonably elevated. Since the relapse persisted andwas severely disabling, it was decided to further administer a secondcourse of a higher dosage of peptide and with a higher frequency, andthe patient received 100 mg pMBP82-98 two times daily for two days (day19 and 20: it#6, it#7, it#8 and it#9). Anti-MBP was measured before and30 minutes after each injection. Subsequent to this increased dosage andfrequency, F anti-MBP was suppressed to negligible levels, and whentested a week later (day 28) his CSF profile was compatible with slowlyprogressing disease (F/B anti-MBP=1.0). At this time the patientreceived a third intravenous injection of 500 mg pMBP 82-98 (IV#3) whichdid not down regulate any more anti-MBP production.

EXAMPLE 6 Intravenous Administration of MBP Peptides in an Attempt toPrevent Future Relapses

Two patients with relapsing-progressive MS, who had frequent relapseswere injected intravenously, with either pMBP86-95 or pMBP82-98 todetermine if this route of administration will prevent further attacks.

The first patient was experiencing 2 to 3 relapses per year for 4 years,with resulting stepwise progression of spastic paraparesis (FIG. 15).She received two intravenous injections 6 months apart, one of 400 mgpMBP86-95 (IV#1) and the second of 400 mg. pMBP82-98 (IV#2); clinicalmonitoring and CSF analysis were performed monthly. FIG. 15 showsanti-MBP levels over a period of 9 months (upper boxed area). The firstintravenous injection down regulated anti-MBP synthesis for about 2months. During the third month post injection, this patient experienceda clinical relapse; unfortunately CSF was not obtained at that time.During the subsequent 2 to 3 months, after the relapse resolved that theillness reentered the chronic progressive phase, this patient receivedthe second intravenous injection (IV#2). CSF anti-MBP levels were againsuppressed for 2 months but, three months after the second injection,the patient had another relapse associated with markedly elevated Fanti-MBP. Similar to the relapse rate she had in the previous 4 years,this patient continued to experience 2 to 3 relapses per year despitereceiving two intravenous injections of pMBP86-95 and pMBP82-98.

A second patient (FIG. 16) who experienced 1 to 4 acute relapses peryear for the previous 10 years (upper scale) became seriously disabled,paraplegic and confined to a wheelchair. During the 11th year thepatient once again experienced four relapses (upper boxed area),although receiving MBP synthetic peptides intrathecally andintravenously. During the first relapse, after receiving intrathecallytwo injections of 50 mg pMBP86-95 on day 1 and day 6 (it#1, it#2) her Fanti-MBP level was substantially reduced; on day 6 she also receivedintravenously 300 mg of pMBP86-95 (IV) which subsequently suppressedboth F and B antibody for the next 3 months. Four months after theintravenous injection, this patient experienced another clinical relapsewhich continued to worsen in time: CSF antibody levels were highlyelevated, and 6.5 months after the IV injections the patient received acourse of four daily injections of 50 mg pMBP82-98 (it#3, it#3, it#5 andit#6), which failed to suppress F antibody levels and to resolve theclinical relapse.

EXAMPLE 7 Comparison of Different Routes of Peptide Administration

In initial studies, synthetic MBP peptides were administered to eightchronic progressive MS patients. Patients received intrathecally eitheran MBP binding peptide MBP(75-95) or a control non-binding peptideMBP(35-58) in increasing doses from 1 to 10 mg in 5 ml of saline; thefour patients who initially received the control non-binding peptide(MBP35-58) later received the binding MBP(75-95) peptide.

Injection of MBP(75-95) into CSF resulted in transient neutralization ofF MBP specific antibodies; bound MBP autoantibodies were not affected.The duration of the effect lasted 1 hour (1 mg of peptide), 24 hours(2.5 mg of peptide) or 7 days (5-10 mg of peptide). Since the effect ofintrathecal peptide administration was incomplete (B anti-MBP remainedelevated) and relatively short-live d, this route of administration wascompared to intravenous injection. In contrast to intrathecaladministration, both free and bound MBP autoantibodies becameundetectable one month after a single intravenous injection of 500 mg ofMBP(75-95) and remained at low levels for three months and after abooster injection for up to 26 months (FIG. 17). Similar observationswere made to date in approximately 70 patients with chronic progressiveMS who were injected intravenously with an MBP binding peptide such asMBP75-95, MBP86-95, MBP82-98. A dose of 500 mg (5 mg/kg bodyweight) in10-50 ml of normal saline, was chosen because of the larger volume ofblood versus CSF (factor 15) and the rapid clearance of peptides fromthe bloodstream through the kidney; peptide doses corresponding to thosegiven intravenously were not administered intrathecally because suchvolumes could not be injected into CSF. In summary, intrathecaladministration, in the dose range tested in these patients, resulted ina transient “mopping” of F anti-MBP only, in contrast to intravenousinjection(s) that down regulated anti-MBP synthesis, a singleintravenous injection induced long-lasting tolerance.

EXAMPLE 8

Duration of Tolerance Following Intravenous Administration of the MBPPeptide

Based on these results, kinetics of tolerance to MBP were examined todate in approximately 70 patients with chronic-progressive MS who werefollowed for over two years following multiple intravenous injections ofMBP(75-95), MBP(86-95) or MBP(82-98). Peptides were dosed at 5-6 mg/kgbody weight (256-500 mg) and injected intravenously in 10-50 ml ofsaline. Prior to intravenous peptide administration, all 13 patients hadhigh levels of free and bound MBP antibodies in CSF (FIG. 18, Table 3).One month following peptide administration, MBP specific antibodiesbecame essentially undetectable and remained at low levels generally for34 months, at which time antibody levels began to rise again; somereturning to their initial levels by 8 months. Six to ten monthsfollowing IV#1, all patients received a booster injection (IV#2) of275-500 mg (5-6 mg/kg body weight) of MBP(82-98) in 10 ml of saline(IV#2). The longer peptide chosen for the second injection was moresoluble and could be dissolved and administered in a smaller volume. Inthis group as a whole, CSF anti-MBP levels declined dramatically within6 weeks to 2 months from the injection and remained undetectable for alonger time (up to 26 months). Of the whole group of approximately 70patients, one was unable to complete the study due to a pulmonaryembolus and subsequent anticoagulant therapy that prevented furtherlumbar punctures, and another was excluded from follow-up because ofreceiving high dose intravenous corticosteroids. Individually, of theapproximately 70 patients, about 63 had undetectable anti-MBP levels,18-26 months after the booster injection.

EXAMPLE 9 Long-Lived Tolerance in Patients with the HLA-DR2 Haplotype

The HLA-DR haplotypes of MS patients were determined by molecular typingof genomic DNA (Table 3). Four of eleven patients who completed thestudy carried the disease associated DR2 haplotype (DRB1*1501 orDRB1*15021); all of these patients had low or undetectableautoantibodies levels one year following the second intravenous MBPpeptide injection. The MBP peptide binds with high affinity to HLA-DR2and is immunodominant for HLA-DR2 restricted, MBP specific T cells.HLA-DR4 (DRB1*0401) and HLA-DR7 (DRB1*0701) bind the MBP peptide thatwas administered; binding studies have not been done for the DRmolecules carried by patient k(M) (DRB1*0407, DRB1*0801). The MBPpeptide is not bound by HLA-DR3 (DRB1*03011); two patients who hadelevated anti-MBP at the end of the study carried the DRB1*03011haplotype (Table 3). These data indicate that the duration of toleranceto MBP depends on the HLA-DR haplotype of a patient. Tolerance may bemore long-lived when both MBP specific T cells and B cells aretolerized. TABLE 3 HLA-DR haplotypes of MS patients Patient HLA-DRHaplotypes Total Anti-MBP (Ru) A. Low levels of total anti-MBP at 1 yearfollowing IV#2 b (F) DRB1*1501 4.1 e (F) DRB1*1501 DRB1*1303 2.5 m (M)DRB1*1501 DRB1*0101 3.9 l (F) DRB1*15021 DRB1*0403 3.9 a (M) DRB1*1401DRB1*0701 4.1 f (F) DRB1*0701 2.4 k (M) DRB1*0407 DRB1*0801 4.5 B.Elevated levels of total anti-MBP at 1 year following IV#2 j (M)DRB1*03011 7.3 h (F) DRB1*0101 DRB1*0701 9.7 g (F) DRB1*0101 DRB1*110119.1 I (M) DRB1*0403 DRB1*03011 19.0total anti-MBP: free anti-MBP + bound anti-MBPHLA-DR haplotypes of 11 MS patients who completed the 1 year follow upform the second intravenous peptide injection (IV#2). All four patientswith HLA-DR2 haplotype (DRB1*1501 or DRB1*15021) had low autoantibodylevels one year following IV#2.

EXAMPLE 10 Subcutaneous Peptide Administration does not Induce Tolerance

The optimal route of peptide administration was further investigated bysubcutaneous injection(s) of MBP(82-98) in saline in a group of 33 MSpatients. In 26 MS patients, increasing amounts (1 to 100 mg) of asingle subcutaneous injection of MBP(82-98) did not affect CSFautoantibody levels to MBP (data not shown); eight of these patientssubsequently received an intravenous peptide injection and within twomonths CSF antibody levels became undetectable (Table 4A). In five otherpatients, a total dose of 900-1000 mg (5×100 mg, daily for fiveconsecutive days, followed by another subcutaneous injection of 400 or500 mg) only resulted in a modest decrease of MBP antibody levels in CSF(Table 4B). To examine whether a different schedule of administrationwould be more effective, two patients received two subcutaneousinjections of 250 mg of MBP(82-98) one month apart (Table 4C). Again,autoantibody levels were not affected. Taken together, these datademonstrate that only intravenous administration of the MBP peptideinduces long-lived tolerance to MBP at the peptide doses tested in thisstudy. TABLE 4 A MBP MBP (82-98) Elapsed (82-98) Patient SC Baseline 6-7weeks time Baseline IV#1 2 months 4 months ID (sex) mg f b f b (months)f b mg f b f b E(F) 5 9.1 11.2 10.2 10.4 6 9.3 9.8 400 1.0 1.1 1.4 1.0K(F) 7 2.1 3.4 3.1 5.9 6.5 6.3 6.7 500 1.5 0.8 N(F) 10 8.1 8.0 7.1 8.1 86.6 5.6 500 3.0 3.0 Q(F) 40 9.9 10.1 10.9 8.3 6 10.0 9.3 400 1.5 1.6 2.12.1 R(M) 50 10.2 10.3 11.1 7.4 6 7.5 9.9 500 1.5 1.6 1.5 1.7 S(F) 60 4.14.3 6.1 5.4 6.5 7.4 7.4 500 1.8 0.9 X(M) 100 9.9 7.3 9.5 8.2 4.5 9.7 9.0500 1.4 1.0 1.5 1.1 Z(M) 100 9.9 8.4 10.9 10.1 4.5 10.5 9.7 500 2.0 1.91.5 1.6 MEAN 7.9 7.9 8.6 8.0 8.4 8.4 1.7 1.5 1.6 1.5 SD 2.9 2.6 2.7 1.71.5 1.5 0.6 0.7 0.3 0.4 B MBP (82- Patient MBP (82-98) Elapsed 98) ID SCBaseline 6-7 weeks time SC 7 weeks (sex) mg f b f b (months) mg f bAA(F) 100/d × 5 7.7 8.1 4.4 4.9 0.5 400 4.3 3.4 BB(F) 100/d × 5 5.4 5.43.5 3.7 0.5 500 2.0 2.5 CC(M) 100/d × 5 5.9 5.4 6.9 8.8 — — DD(F) 100/d× 5 4.6 4.8 2.7 1.9 0.5 500 3.0 2.8 EE(F) 100/d × 5 7.4 8.9 3.7 3.9 0.5400 2.6 2.4 MEAN 6.2 6.5 4.2 4.6 3.0 2.9 SD 1.2 1.7 1.4 2.3 0.8 0.7 CPatient MBP (82-98) ID SC Baseline 15 weeks (sex) mg f b f b GG(M) 250/m× 2 8.4 8.7 7.1 8.3 FF(F) 250/m × 2 4.8 5.3 5.4 4.2A. Eight patients received a single subcutaneous injection of MBP(82-98)(5-100 mg in 1-5 ml saline) which had no effect on MBP autoantibodylevels. In contrast, a single intravenous injection (400-500 mg) of thesame peptide administered 4.5 to 8 months later resulted in undetectableCSF autoantibody levels.B. Repeated subcutaneous injections of high doses of MBP(82-98) (100mg/day for five consecutive days) had a modest effect on CSF anti-MBPlevels; an additional high dose (400 or 500 mg) of MBP(82-98)administered subcutaneously two weeks after the first set of injectionsdid not further reduce autoantibody levels.C.Two subcutaneous injections of high doses of MBP(82-98) (2×250 mg, onemonth interval) had no effect on MBP autoantibodies in CSF. Takentogether, these data demonstrate that only the intravenous route ofadministration is effective in inducing tolerance to MBP.

Various modifications may be made to the preferred embodiments withoutdeparting from the spirit and scope of the invention as defined in theappended claims.

1. A peptide of the formula: Asp Glu Asn Pro Val Val His Phe Phe Lys AsnIle Val Thr Pro Arg Thr; including substitutions, additions or deletionsthereof, provided that said substitutions, additions or deletionsprovide a peptide that is capable of neutralizing or modulating theproduction of anti-myelin basic protein.
 2. A pharmaceutical compositioncontaining as an active ingredient a peptide of the formula: Asp Glu AsnPro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr; includingsubstitutions, additions or deletions thereof, provided that saidsubstitutions, additions or deletions provide a peptide that is capableof neutralizing or modulating the production of anti-myelin basicprotein, in admixture with a pharmaceutical acceptable carrier.
 3. Amethod of treating multiple sclerosis in a patient in need thereof byadministering to said patient an effective amount of a peptide of theformula: Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro ArgThr; including substitutions, additions or deletions thereof, providedthat said substitutions, additions or deletions provide a peptide thatis capable of neutralizing or modulating the production of anti-myelinbasic protein, in admixture with a pharmaceutical acceptable carrier. 4.A method of treating multiple sclerosis in a patient in need thereof byadministering to said patient an effective amount of a peptide of theformula: R₁-Val-His-Phe-Phe-Lys-Asn-Ile-R₁

and salts thereof, wherein R₁ and R₂ are independently selected from thegroup consisting of hydrogen, hydroxy, an amino acid residue and apolypeptide residue; provided that R₁ and R₂ are not both hydrogen orhydroxyl at the same time; including substitutions, additions ordeletions thereof provided that said peptide is capable of neutralizingor modulating the production of anti-myelin basic protein, alone or incombination, in admixture with a pharmaceutical acceptable carrier;wherein said method comprises administering sequential doses of saidpeptide.
 5. The method of claim 4, wherein R₁ is Asn-Pro-Val- and R₂ ishydrogen or hydroxy.
 6. The method of claim 4, wherein R₁ is Pro-Val-and R₂ is -Val.
 7. The method of claim 4, wherein R₁ is Val- and R₂ is-Val-Thr.
 8. The method of claim 4, wherein R₁ is hydrogen or hydroxyand R₂ is -Val-Thr-Pro.
 9. The method of claim 4, wherein R₁ isLys-Ser-His-Gly-Arg-Thr-Gln-Asp-Glu-Asn-Pro-Val- and R₂ is -Val-Thr. 10.The method of claim 4, wherein R₁ is Asp-Glu-Asn-Pro-Val- and R₂ is-Val-Thr-Pro-Arg-Thr.
 11. The method of claim 4, wherein the peptide isadministered intravenously, intrathecally or a combination of both. 12.The method of claim 11, wherein the peptide is administeredintravenously at a dose ranging from 1 mg/kg of body weight to 10 mg/kgof body weight.
 13. The method of claim 11, wherein the peptide isadministered intrathecally at a dose ranging from 1 mg to 100 mg. 14.The method of claim 11, wherein the peptide is administered at leastdaily for four to five days.
 15. The method of claim 14, wherein thepeptide if further administered in an additional dose about one weekafter the first injections.